Mechanism of Transcription Termination by E. coli RNA Po

大肠杆菌 RNA Po 转录终止机制

• 批准号: 6763575
• 负责人: MIKHAIL KASHLEV
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6763575
• 关键词: DNA directed RNA polymerase; Escherichia coli; bacterial genetics; bacterial proteins; chemical association; chemical stability; enzyme activity; microorganism culture; transcription factor; transcription termination

This project concerns the investigation of the mechanism of transcription termination in Escherichia coli and the yeast S. cerevisiae in the highly purified in vitro transcription system. This system involves the E. coli RNA polymerase [hexahistidine-tagged at the carboy-terminus of beta' subunit (rpoC)] and the yeast RNA polymerase II (Pol II, hexahistidine-tagged at N-terminus of RPB3 subunit) core enzyme immobilized in a solid phase to which synthetic DNA and RNA components were added to reconstruct the authentic elongation complex. The elongation complex represents a form of RNA polymerase traveling along the gene and synthesizing the RNA transcript. Normally, the elongation complex is highly stable and grips the DNA and the RNA very tightly. However, when RNA polymerase reaches the sequence called transcription terminator, the "grip" is relieved and the complex falls apart momentarily. The terminator consists of two elements: the stable hairpin-like structure formed in the RNA behind RNA polymerase, and the oligo-uridine track located next to the hairpin in the RNA. The mechanism, that causes RNA polymerase dissociation at terminator is unknown. It's believed, that the strength with which the end of the RNA is hybridized to the DNA in the moving polymerase is a major factor of the elongation complex stability. When the hybrid is weak or short the complex spontaneously dissociates. One of the ideas how the hairpin might work is that it reduces the RNA:DNA hybrid in the polymerase beyond its normal length. We addressed the role of the RNA hairpin in destabilization of elongation complex by performing the statistical measurements of the dissociation kinetics of elongation complexes at different variants of transcription terminator. The project involved four consecutive steps. First, the elongation complex was reconstituted followed by the analyses of its stability using the salt-sensitivity assay. The conclusions we have reached can be summaried as follows: (i) We show that folding of the hairpin disrupts the three upstream base pairs of the 8-bp RNA:DNA hybrid, a major stability determinant in the elongation complex. During termination, the hairpin does not directly compete for base pairing with the 8-bp hybrid. Thus, melting of the hybrid results from spatial restrictions in RNA polymerase that couple the hairpin formation with the disruption of the hybrid immediately downstream from the stem. Shortening the weak rU:dA hybrid from 8 nt to 5 nt causes dissociation of the complex. (ii) We demonstrate that a similar mechanism, involving melting of 8-bp RNA:DNA hybrid by the hairpin, disrupts elongation complexes of yeast RNA polymerase II in vitro. Thus, melting of the RNA:DNA hybrid arises as a general mechanism inducing RNA polymerase release in prokaryotes and eukaryotes.

该项目涉及对大肠杆菌中转录终止机理的研究，而在高度纯化的体外转录系统中，酿酒酵母的调查。 This system involves the E. coli RNA polymerase [hexahistidine-tagged at the carboy-terminus of beta' subunit (rpoC)] and the yeast RNA polymerase II (Pol II, hexahistidine-tagged at N-terminus of RPB3 subunit) core enzyme immobilized in a solid phase to which synthetic DNA and RNA components were added to reconstruct the真实的伸长综合体。伸长络合物代表RNA聚合酶沿基因行进并合成RNA转录本的一种形式。通常，伸长复合物高度稳定，并非常紧密地抓住DNA和RNA。但是，当RNA聚合酶达到称为转录终结剂的序列时，“握持”就会缓解，复合物会瞬间散开。终结子由两个元素组成：在RNA聚合酶后面的RNA中形成的稳定发夹状结构，以及位于RNA中发夹旁边的寡尿尿道轨道。导致RNA聚合酶在终结剂处的RNA聚合酶解离的机制尚不清楚。人们认为，将RNA末端与运动聚合酶中的DNA杂交的强度是伸长复合物稳定性的主要因素。当杂种较弱或短短复合物自发解离时。发夹的工作方式之一是，它可以降低RNA：聚合酶中的DNA杂种超出其正常长度。我们通过在转录终止剂的不同变体上对伸长式复合物的分离动力学的统计测量来解决RNA发夹在伸长复合物的不稳定中的作用。该项目涉及连续四个步骤。首先，重组伸长络合物，然后使用盐敏化测定法对其稳定性进行分析。我们得出的结论可以总结如下： （i）我们表明，发夹的折叠破坏了8 bp RNA的三个上游底座对：DNA混合动力，这是伸长仪中的主要稳定性决定因素。在终止期间，发夹不会直接竞争与8 bp混合动力车的基础配对。因此，杂种的融化是由RNA聚合酶中的空间限制造成的，将发夹的形成与立即从茎下游下游的杂种造成的破坏。缩短弱的RU：DA杂交从8 nt到5 nt会导致复合物解离。 （ii）我们证明了一种类似的机制，即涉及8 bp RNA的熔化：发夹的DNA杂交，在体外破坏了酵母RNA聚合酶II的伸长复合物。 因此，RNA：DNA杂交的熔化是作为诱导原核生物和真核生物中RNA聚合酶释放的一般机制而产生的。